IDENTIFICATION OF THE HORSE EPIDERMAL GROWTH-FACTOR (EGF) CODING SEQUENCE AND ITS USE IN MONITORING EGF GENE-EXPRESSION IN THE ENDOMETRIUM OF THE PREGNANT MARE
F. Stewart et al., IDENTIFICATION OF THE HORSE EPIDERMAL GROWTH-FACTOR (EGF) CODING SEQUENCE AND ITS USE IN MONITORING EGF GENE-EXPRESSION IN THE ENDOMETRIUM OF THE PREGNANT MARE, Journal of molecular endocrinology, 12(3), 1994, pp. 341-350
The PCR technique and highly degenerate oligonucleotide primers were u
sed to amplify a 282 bp fragment of the horse (Equus caballus) epiderm
al growth factor (EGF) cDNA. The clone corresponded to 94 amino acids
of the EGF precursor molecule. The deduced amino acid sequence of the
53 residue EGF mitogenic peptide within the precursor sequence showed
60-70% identity with five other published EGF sequences. The PCR cDNA
fragment hybridized to a 4.9 kb transcript in horse kidney and endomet
rial RNA which was of a similar size to the mature EGF transcript foun
d in other mammalian species. The horse cDNA clone was used in Norther
n blots to monitor EGF expression in the endometrium of pregnant mares
up to day 83 of gestation (term = 330-340 days). The level of express
ion increased from day 33 and showed a further dramatic increase betwe
en days 35 and 45, which coincides with the onset of implantation and
placentation in this species. Levels remained elevated up to day 83. T
he horse DNA sequence was used to design sense and antisense oligonucl
eotide probes (45-mers) for in situ hybridization studies. The antisen
se probe showed specific hybridization to the glandular, but not lumen
al, epithelial cells of the endometrium and there was no signal in fet
al membranes. The in situ hybridization signal increased between days
35 and 45 to a similar degree to that observed in the Northern blot an
alysis. This dramatic increase in EGF expression in the glandular epit
helium of the mare's endometrium during pregnancy may provide a mitoge
nic stimulus to the endometrium and/or trophoblast to facilitate place
ntal differentiation and attachment. Alternatively, the precursor coul
d be involved in the endometrial gland secretory process which is nece
ssary to produce uterine milk for fetal sustenance. The PCR cloning me
thods used in this study should be generally applicable to the cloning
of EGF cDNAs from other species.